AIMS: Compound K (C-K; 20-O-D-glucopyranosyl-20(S)-protopanaxadiol) is a novel ginsenosides metabolite formed by intestinal bacteria and does not occur naturally in ginseng. In this study, we investigated whether administration of C-K has protective effects on myocardial ischemia-reperfusion injury and its potential mechanisms. MAIN METHODS: We used in vivo mouse models of ischemia-reperfusion injury and performed biochemical assays in excised hearts. KEY FINDINGS: C-K reduced infarct size compared with the control group after ischemia-reperfusion. Immunoblot analysis showed that C-K significantly enhanced protein kinase B (Akt) and endothelial nitric oxide synthase (eNOS) activity. Wortmannin, a phosphoinositide 3-kinase (PI3K) inhibitor, blocked cardiac protection in vivo and attenuated phosphorylation of Akt and eNOS. Additionally, hearts of C-K pretreated mice showed inhibition of mitochondrial swelling induced by Ca(2+). SIGNIFICANCE: This study showed that Compound K pretreatment has protective effects on myocardial ischemia-reperfusion injury, partly by mediating the activation of PI3K pathway and phosphorylation of Akt and eNOS.